This invention relates to DNA for transforming cells to enable regulated in vivo protein formation, to the cells that are used in the transformation, to precursors for making such transforming DNA, and to methods of using the DNA.
It is often useful to engineer an organism with genetic information whose expression is subject to in vivo control. For example, when using engineered organisms to produce a desired compound, it is desirable to control expression of relevant genes (genes coding for the compound itself, for a compound precursor, or for enzymes in the compound's synthetic pathway) so that compound production is reduced or prevented during the organism's exponential growth phase, because compound production may be deleterious to the organism or may slow its growth due to energy demands. By controlling compound production in the growth phase, the engineered organism may grow more rapidly and may not be at a competitive disadvantage with respect to nonproducing organisms (e.g. mutants). Once a satisfactory population of engineered organisms is obtained, they must be made capable of product manufacture. Preferably, such regulatory control is strong; that is, compound production should be dramatically reduced or eliminated when desired, and compound production should be as copious as possible when desired. Apart from compound biosynthesis it may be desirable to control genetic expression to control other traits of engineered organisms.
Some gene promoters are influenced by regulatory effector molecules which interact with sites at or near the promoter to enable or prevent expression of structural genes under the promoter's control. Thus, expression of a structural gene that has been introduced to an organism may be influenced by engineering the gene to be controlled by such a regulated promoter and then using the regulatory effector molecule to control the gene's expression [Backman et al. (1976) Proc. Nat'l Acad. Sci. USA 71: 4174.].
Other gene promoters are essentially unregulated. These promoters tend to be much stronger (i.e., they induce greater expression of structural genes under their control) than regulated promoters. Until now, however, very strong unregulated promoters have been difficult to use because they cannot be cloned unless followed by an effective signal for termination of transcription and because regulation may be desirable for the reasons discussed above.
Finally, it is known that in certain systems, a DNA segment reversibly inverts, creating a population of organisms that is heterogenous in that in some organisms the DNA segment is in one orientation and in others it is in the opposite orientation. For example, Silverman et al. report an analysis of the genes involved in the DNA inversion responsible for oscillation of flagellar phenotypes of Salmonella, ["Analysis of the Functional Components of the Phase Variation System", Cold Spring Harbor Symposium on Quantitative Biology, 45: 17-26, (Cold Spring Harbor Laboratory, New York, (1981)]. Silverman et al. disclose that the Salmonella DNA inversion is a site-specific recombination event involving 14 bp inverted repeat sequences.
Iino et al. disclose that certain bacteriophage (Pl and Mu) express a DNA inversion factor which is effective to increase the frequency of flagellar phase variation in Salmonella by increasing reversible DNA inversion in that organism, "Trans-acting Genes of Bacteriophages of Pl and Mu Mediate Inversion of a Specific DNA Segment Involved in Flagellar Phase Variation of Salmonella", Cold Spring Harbor Symposium on Quantitative Biology, 45:11-16, (Cold Spring Laboratory New York, 1981)].
Struhl (1981) J. Mol. Biol. 152:517-533 disclose that the bacteriophage lambda int gene mediates many events including deletion events which randomly generate new gene sequences some small fraction of which may have genetic activity.